Inhibitors of angiotensin converting enzyme

ABSTRACT

The object of the present invention is to provide a peptide having angiotensin converting enzyme inhibitory activity also improved in the taste, and to provide a method of producing an angiotensin converting enzyme inhibitors comprising at least one of such enzymes. Another object of the present invention is to provide an antihypertensive comprising a peptide having angiotensin converting enzyme inhibitory activity. Thus, the present invention is any of the five peptides represented by the following formula (1) to (5) and the salts thereof, soybean protein hydrolysates containing these peptides and the method of producing these peptides or the salts thereof comprising hydrolyzing soybean proteins with protease D3:(1)Tyr-Val-Val-Phe-Lys; (2)Pro-Asn-Asn-Lys-Pro-Phe-Gln; (3)Asn-Trp-Gly-Pro-Leu-Val; (4)Ile-Pro-Pro-Gly-Val-Pro-Tyr-Trp-Thr; (5)Thr-Pro-Arg-Val-Phe.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to an angiotensin converting enzymeinhibitor, particularly an inhibitor peptide for angiotensin convertingenzyme and a method of producing the inhibitor. More specifically, thepresent invention relates to an inhibitory peptide for angiotensinconverting enzyme which is capable of being orally ingested and a methodof producing the peptide. The present invention further relates to anantihypertensive containing an angiotensin converting enzyme inhibitor,particularly an inhibitory peptide for angiotensin converting enzyme.

[0002] Angiotensin converting enzyme generates angiotensin II havingvasopressor activity including vasoconstriction by cleaving C-terminalHis-Leu of inactive angiotensin I. It is a vasopressor enzyme alsohaving the activity of degrading bradykinin which has strong vasodilatoraction. It is known that hypertension can be treated by inhibiting thefunction of angiotensin converting enzyme. An angiotensin convertingenzyme inhibitor was originally found in a snake poison as a peptidewhich enhances ileum smooth muscle contraction in guinea pig caused bybradykinin and was called as bradykinin potentiator.

[0003] In the later studies, it was elucidated that a fraction havingangiotensin converting enzyme inhibitor activity existed in thedegradation product of food proteins such as pepsin hydrolysates ofsoybean proteins or ginseng proteins. As such an example, for example, apeptide having hypotensive function was found among the peptidefragments in human β casein. Furthermore, angiotensin converting enzymeinhibitory peptides are also found among the hydrolysates which had beenproduced with proteolytic enzymes. Such peptides together with theirsequences are reported in, for example, Publication of UnexaminedJapanese Patent Application (JP-Kokai) No. 8-225593 (pepsin degradationproducts of soybean proteins), JP-Kokai No. 8-269088 (pepsin degradationproducts of K casein glycomacropeptide), JP-Kokai No. 11-29594(thermoase PC-10 degradation product of tuna fish meat), JP-Kokai No.11-335393 (pepsin degradation products of ginseng) and JP-Kokai No.2000-4799 (endo-proteinase and exo-proteinase degradation products ofpotato protein). The angiotensin converting enzyme inhibitory peptideswhich have been reported thus far generally tend to contain Pro residuesand are frequently the peptides having high hydrophobicity.

[0004] On the other hand, independent of these studies, variousproteolytic enzymes are used to modify the properties of proteins forfood products. For example, the applicant reported a thiol proteasehaving high proteolytic activity toward proteins, particularly proteinsfor food products (JP-Kokai No. 8-264) and the applicant also reportedthat the hydrolysates having low bitterness were prepared with theprotease (protease D3) on proteins (JP-Kokai NO. 12-83695).

SUMMARY OF THE INVENTION

[0005] It has been literary reported that the substances havingangiotensin converting enzyme inhibitory activity, particularly thepeptides having angiotensin converting enzyme inhibitory activity,existed in the degradation products of proteins for foods, as describedabove. However, the enzymatic hydrolysates disclosed in these referencesare produced with protease formulations such as pepsin or papain, andare known as having strong bitterness. Thus, orally ingesting thesehydrolysates as the product containing angiotensin converting enzymeinhibitor peptides will be limited in the light of their taste.

[0006] Accordingly, the object of the present invention is to provide apeptide having an angiotensin converting enzyme inhibitory activity withimproved in the taste, and to provide a method of producing anangiotensin converting enzyme inhibitors comprising at least one of suchpeptides.

[0007] Another object of the present invention is to provide anantihypertensive comprising the peptide having angiotensin convertingenzyme inhibitory activity.

[0008] The inventors of the present invention have found that theprotease D3 derived from germinating soybean cotyledons is an enzymewhich can produce hydrolysates having less bitterness than thoseproduced with other commercially available enzymes (JP-Kokai No.2000-83695) and that angiotensin converting enzyme inhibitor existed inthe soy protein hydrolysates with D3. Further investigation of thehydrolysates led to the establishment of the present invention.

[0009] Accordingly, the peptide of the present invention which has anangiotensin converting enzyme inhibitory activity and which is improvedin the taste is any of the five peptides represented by the followingformula (1) to (5) and the salts thereof:

[0010] (1) Tyr-Val-Val-Phe-Lys (SEQ ID NO: 1),

[0011] (2) Pro-Asn-Asn-Lys-Pro-Phe-Gln (SEQ ID NO: 2),

[0012] (3) Asn-Trp-Gly-Pro-Leu-Val (SEQ ID NO: 3),

[0013] (4) lle-Pro-Pro-Gly-Val-Pro-Tyr-Trp-Thr (SEQ ID NO: 4),

[0014] (5) Thr-Pro-Arg-Val-Phe (SEQ ID NO: 5),

[0015] wherein Tyr represents tyrosine, Val represents valine, Pherepresents phenylalanine, Lys represents lysine, Pro represents Proline,Asn represents asparagine, Gln represents glutamine, Trp representstryptophan, Gly represents Glycine, Leu represents leucine, Ilerepresents isoleucine, Thr represents threonine, Arg representsarginine, respectively.

[0016] The method according to the present invention is the method ofproducing hydrolysates characterized in that a protein, particularly asoybean protein, is reacted with protease D3.

[0017] The present invention is also an antihypertensive comprising atleast one of these peptides or the salts thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is the schematic diagram showing the procedures ofconstructing plasmid pUCTRPproD3-N. pUCTRPproD3-N contains the promoterregion derived from pUCTRPMTG(+)D2trp and the sequence encoding thepro-part of D3.

[0019]FIG. 2 is the schematic diagram showing the procedures ofconstructing plasmid pUC-D3-C. pUC-D3-C contains the sequence encodingthe sequence of the pro-part and mature D3 sequence.

[0020]FIG. 3 is the schematic diagram showing the procedures ofconstructing plasmid pUCTRPproD3. pUCTRPproD3 contains the promoterregion derived from pUCTRPMTG(+)D2trp, the sequence encoding thepro-sequence of D3 and mature D3 sequence.

[0021]FIG. 4 shows the chromatography on gel filtration and angiotensinconverting-enzyme inhibitory activity of soybean protein hydrolysateswith protease D3. Open square (□) represents the absorbance at 215 nmand black circle () represents the inhibition rate (%) for the enzyme.

[0022]FIG. 5 shows the chromatography on reverse-phase columnchromatography and angiotensin converting enzyme inhibitory activity ofthe fractions that exhibited angiotensin converting enzyme inhibitoryactivity after gel-filtration of soybean protein hydrolysates solutionwith protease D3. Open square (□) represents the absorbance at 215 nmand black circle () represents the inhibition rate (%) for the enzyme.

[0023]FIG. 6 shows the hypotensive effect of the soybean proteinhydrolysates solution prepared with protease D3. Black diamondrepresents the change in the blood pressure in SHR rats for the case inwhich the control solution was given, black rectangular (▪), blacktriangle (▾), cross mark (X) and black circle () represent the case inwhich 100 mg/kg, 500 mg/kg and 1000 mg/kg of soybean proteinhydrolysates prepared with D3 was given, respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The hydrolysates containing the peptides of the present inventionmay be easily prepared in large quantity with protease D3.

[0025] Protease D3 used for producing hydrolysates containing thepeptides of the present invention is the proteolytic enzyme which allowsthe production of peptides having low bitterness, as described above.Protease D3 may be obtained from, for example, germinating soybeancotyledons at about day 10 of germination according to the methoddescribed in JP-Kokai No. 8-264. Alternately, recombinant protease D3(rD3) can be also obtained by autocatalytic processing of recombinantinactive protease proD3 having pro-sequence. Recombinant proD3 may beproduce in, for example, E.coli, yeast or Aspergillus. Protease D3derived from germinating soybean cotyledons is described in JP-Kokai No.9-121870. In JP- Kokai No. 9-121870, D3-α and D3-β are described asrecombinant D3s, both of which may be used for producing the peptides ofthe present invention.

[0026] More specifically, rD3 may be produced by using trp promoter, asthe promoter which can be easily induced by Tryptophan defective mediumand the clone pUCa (JP-Kokai No. 9-12-121870, FERM NP-7835 (originallydeposited as FERM P-14687) (Independent Administrative Agency, NationalInstitute of Advance Industrial Science and Technology, Tsukuba Central6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566 Japan)).Optionally, it is possible to modify the sequence of promoter region orof the further upstream region or to change the DNA sequence of D3 genetaking the codon usage into account.

[0027] Trp promoter is a strong promoter. For example, plasmid pTTG2-22(JP-Kokai No.6-225775), which highly expressed Crysophrys majortransglutaminase (TG) gene, used trp promoter where the upstream regionof Crysophrys major TG was designed for high level expression ofheterologous proteins in E.coli. Additionally, plasmid pUCTRPMTG(+)D2(JP-Kokai No. 11-75876), which highly expressed microbialtransglutaminase (MTG) gene, used the upstream region containing trppromoter of this Crysophiys major TG expression plasmid, and the regionwas introduced into multi-copy plasmid pUC19 to highly express MTG. Forthe purpose of producing proD3 in the present invention, the promoterregions of these plasmids are suitable.

[0028] The conversion into high-active D3 by autocatalytic activationmay be conducted in the condition described in JP-Kokai No. 9-121870,namely by subjecting proD3 to 200 mM NaCI solution at about pH 4, andincubating at between about 30° C. and about 50° C., preferably atbetween about 35° C. and about 45° C. This enzyme may be reacted withproteins at pH 3 to pH 7, preferably at pH 3.5 to 5.5, at about 30° C.to 50° C., preferably at about 35° C. to 45° C. Protease D3 canhydrolyze proteins very efficiently at these conditions to generate thepeptides of the present invention.

[0029] The peptides of the present invention may be produced with D3 onsoybean proteins, particularly soybean storage proteins. The substratesfor protease D3 may be purified soybean proteins, but the productsobtained by merely grinding or suspending proteins, optionallyprocessing proteins with acids or alkali or those followed by removingthe insoluble fractions by centrifugation or filtration can also beused. The degradation may be conducted by adjusting the substratesolution such as those described above to about pH 3-7, preferably aboutpH 3.5-5.5, particularly preferably about pH 4.0-pH 5.0, adding to thesolution rD3 prepared as described above such that the ratio ofsubstrate to enzyme (substrate/enzyme) will be about 100/1-1000/1 andincubating for 6-60 hours at about 30° C. to about 50° C., preferably atabout 35° C. to about 45° C. After incubating, the supernatant can beobtained by, for example, centrifugation after inactivating the enzymeby, for example, heating. The supernatant may be neutralized with, forexample, NaOH, and recovering the hydrolysates by, for example,freeze-drying, to obtain crude hydrolysates containing the peptides ofthe present invention. The crude proteolytic degradation productcontaining the peptides of the present invention may be furtherpurified. For purification, the fact that the peptides of the presentinvention have the molecular weight raging 654 to 1029 may be utilized.For example, the high molecular weight fraction having the molecularweight of 2000 or more may be removed by gel filtration chromatographyand the low molecular weight fraction may be further fractionated byreverse-phase chromatography. The angiotensin converting enzymeinhibitory activity in these fractions may be confirmed as follows.Briefly, substances which may be the substrate of angiotensin convertingenzyme such as p-hydroxyl benzoyl glycyl-L-histidyl-L-leucine, anangiotensin converting enzyme such as the commercially available rabbitpulmonal angiotensin converting enzyme and the aforementioned partiallypurified fraction to give rise to the reaction and determined theactivity by colorimetric analysis of the reaction solution.

[0030] In one embodiment of the present invention, the peptides of thepresent invention are obtained by further purifying the hydrolysatesobtained by the methods such as the aforementioned method or by furtherpurifying the partially purified fractions of the hydrolysates. Thetechniques for purifying proteins are well known, which includeion-exchange chromatography, reverse phase high-performance liquidchromatography, affinity chromatography and the like.

[0031] In another embodiment of the present invention, the peptides ofthe present invention are chemically synthesized. The peptides of thepresent invention can be synthesized according to the conventionalpeptide synthesizing methods such as solid phase synthesis. The thussynthesized peptides may be purified by, for example, ion-exchangechromatography, reverse phase high-performance liquid chromatography,affinity chromatography and the like. Such solid phase synthesistechniques and the subsequent purification of peptides are well known inthe art.

[0032] Thus prepared angiotensin converting enzyme inhibitor peptidesmay be used in any type of food products, beverages or medical diet.Additionally, the hydrolysates prepared according to the methods of thepresent invention may be used as the materials for foods after beingappropriately processed dependent on the purpose. The angiotensinconverting enzyme inhibitory peptides of the present invention may beused as antihypertensive. The peptides or the hydrolysates of thepresent invention, alone or together with various pharmaceuticallyacceptable carriers or additives, may be orally or parenterallyadministered, but it is preferable to administer them orally, and mostpreferably they will be administered by mixing them into foods orbeverages as described above. The suitable dosage for aforementionedpurpose may be determined depending on the intended therapeutic effects,the manner of administration, the duration of therapy, the age and thebody weight. For oral administration, 100 mg/(kg body weight) to 1000mg/(kg body weight) or more is preferable as one dosage. The angiotensinconverting enzyme inhibitory peptides of the present invention has noadverse effects or has minimum adverse effects, if any, and they can betherefore ingested more than 1000 mg/kg body weight or ingested pluraltimes a day with foods or beverages over a long period. The othermethods for oral administration or the formulations for parenteraladministration may be obtained according to the conventional formulationmethods depending on the purpose and the manner of administration bymixing with, for example, inert diluents such as lactose, calciumcarbonate or calcium phosphate, swelling agents such as alginic acid,cornstarch or starch, sweetening agents such as sucrose, lactose orsaccharine, aromatics, coloring agents, humectants such as magnesiumstearate and talc or excipients such as wax.

EXAMPLES Reference Example 1 Generation of the Genetic Construct forExpressing proD3

[0033] A part of D3-β cDNA (SEQ ID NO: 6), which had been cloned fromcDNA library prepared with mRNA obtained from germinating soybeancotyledons, was introduced into an expression vector being capable offunctioning in E.coli.

[0034] (1) Ligation of Trp Promoter and D3 Gene

[0035] For D3 gene, the clone pUCa (JP-Kokai No. 9-121870, FERM BP-7835(originally deposited as P-14687)) was used wherein the full length D3-βcDNA had been integrated.

[0036] The integration of DNA fragment was conducted by PCR to ligatingtrp promoter to the upstream region of D3-β cDNA. Firstly, the regioncontaining trp promoter (SEQ ID NO: 8) from MTG expression plasmidpUCTRPMTG(+)D2 (JP-Kokai No.11-75876) and the partial region of the D3cDNA from pUCa were amplified by PCR. The primers for amplifying trppromoter were TRP-N2 (SEQ ID NO: 9) and TRP-C2 (SEQ ID NO: 10) and theprimers for partially amplifying D3 were D3-N (SEQ ID NO: 11) and D3-C(SEQ ID NO: 12), wherein TRP-N2 and D3-N were sense primers, and TRP-C2and D3-C were antisense primers. D3-N was designed such that it couldconnect 11 bases sequence for ligation with the sequence containing trppromoter and the initiating ATG codon to the sequence starting at TACencoding Tyr at position -107 (supposed to be the starting point ofpro-sequence) of D3 indicated as SEQ ID NO: 6. This sequence iscomplementary to the sequence in TRP-C2. Hindlil site was introducedinto D3-C.

[0037] Firstly, PCR was conducted for pUCTRPMTG(+)D2 using the primerTRP-N2 and TRP-C2, and for pUCa using the primer D3-N and D3-C, underthe condition of 35 cycles of [94° C. for 30 seconds, 72° C. for 2minutes], respectively. Each PCR product was extracted withphenol/chloroform, precipitated with ethanol and was dissolved in 100 μlof H₂O.

[0038] 1 μl from each PCR product was taken and mixed together, heatdenatured at 94° C., and was subjected to PCR under the condition of 35cycles of [94° C. for 30 seconds, 55° C. for 1 minute and 72° C for 2minutes] using the primer TRP-N2 and D3-C.

[0039] The product of the second PCR was extracted withphenol/chloroform, precipitated with ethanol, digested with HindIII andEcoRI, and subcloned into pUC 18 to obtain pUCTRPproD3-N (FIG. 1), thesequence of which was confirmed.

[0040] (2) Introduction of Stop Codon Into the C-terminal Sequence PCRwas conducted to introduce the stop codon at the C-terminal of D3-β. Thesense primer D3-N2 (SEQ ID NO: 13) and the antisense primer D3-C2 (SEQID NO: 14) were used. D3-C2 is the primer for mutating TGT encoding Cysof residue No. 248 in the amino acid sequence indicated as SEQ ID NO: 6to TGA, the stop codon.

[0041] PCR was conducted for pUCa using the primers D3-N2 and D3-C2under the condition of 35 cycles of [94° C. for 30 seconds, 55° C. for 1minute and 72° C. for 3 minutes]. Each PCR product was extracted withphenol/chloroform and was cloned into Smal site of pUC18 to obtainpUC-D3-C (FIG. 2), of which sequence was confirmed.

[0042] <Sequence Listing Free Text>

[0043] SEQ ID NO: 8: promoter region of pUCTRPMTG(+)D2

[0044] SEQ ID NO: 9 and SEQ ID NO: 10: PCR primer for amplifying trppromoter region

[0045] SEQ ID NO: 1 1 and SEQ ID NO: 12: PCR primer for amplifying D3region

[0046] SEQ ID NO: 13 and SEQ ID NO: 14: PCR primer for ligating trppromoter region to D3 region

[0047] (3) Construction of ProD3 Expression Plasmid

[0048] ProD3 expression plasmid driven by trp promoter was generated byusing the partial fragment of thus obtained plasmid. Namely,Sacll-HindIII fragment (large) of pUCTRPproD3-N and SaciI-HindIIIfragment (small) of pUC-D3-C were ligated to obtain pUCTRPproD3 (FIG.3).

Reference Example 2 Expression of ProD3 and Production of Mature D3

[0049] The proD3 expression plasmid obtained according to ReferenceExample 1 was expressed in E.coli to finally obtain D3 in its activeform. (1) E.coli strain JM109 (Takarashuzo CO., Ltd.) was transformedwith pUCTRPproD3 and was selected on agar plates containing ampicillinto obtain transformants. The transformants were inoculated onM9-casamino acid medium containing ampicillin and cultured at 37° C. forabout 20 hours. Consequently, the intended D3 gene product wasaccumulated as protein inclusion bodies within the cells.

[0050] After harvesting, the cells were sonicated and the proteininclusion bodies were recovered by centrifugation. The protein inclusionbodies were washed and dissolved in 8M urea—10 mM dithiothreitol—50OmMNaCI—50 mM Tris-HCI—5 mM disodium ethylenediaminetetraacetate solution(pH 8). The resulted solution was also referred to as “solubilized proD3solution”. The three-dimensional structural reconversion (refolding) ofthe denatured proD3 in the solubilized proD3 solution into proD3 havingthe naturally occurring three-dimensional structure was carried outaccording to the following procedures.

[0051] 2.5 ml of solubilized proD3 solution was added to PD-10 column(Amersham Bioscience), which had been previously equilibrated with 1 mMreduced glutathione—0.1 to 0.5 mM oxidized glutathione—50 mM potassiumphosphate—5 mM disodium ethylenediaminetetraacetate solution (pH 10.5)and then 3.5 ml of 50 mM potassium phosphate (pH 10.5) was added. Theeluent was recovered as refolded proD3.

[0052] (2) Autocatalytic Activation

[0053] The solution of refolded proD3 was incubated at 37° C. and at pHof about 4.5. As a result, proD3 having the molecular weight of about 41k was converted to highly active D3 having the molecular weight of about30 k. This highly active D3 was used for proteolytic hydrolysis.

Example 1 Preparation of D3-Degradation Products

[0054] The isolated soybean protein solution was adjusted to about pH4.5 with hydrochloric acid. D3 was added to the isolated soybean proteinsolution such that the ratio substrate/enzyme=500/1 and allow thereaction at 37°C. for 24 hours. After the reaction was completed, theenzyme was inactivated by heating and the supernatant was obtained bycentrifugation. The pH of the supernatant was neutralized to around theneutral point with NaOH and the hydrolysates were obtained throughfreeze-drying.

Example 2 Identification of the Candidates of Angiotensin ConvertingEnzyme Inhibitory Peptides

[0055] The peptides prepared as described in Example 1 were dissolved tothe concentration of 5 mg/ml, subjected to gel filtration column(Superdex75 HR 10/30; Amersham Bioscience) after filtration, and thefractions having the molecular weight of 2000 or less were divided toseven fractions. 0.05% TFA was used as a mobile phase, and the flow ratewas 0.5 ml/min. and the detection was carried out by determining theabsorbance at 215nm. The angiotensin converting enzyme inhibitoryactivity of each fraction was determined and five fractions, G1 to G5(FIG. 4; G 1 fraction: fractions No.10 to No.11; G2 fraction: fractionsNo.12 and 13; G3 fraction: fractions No.14 and 15; G4 fraction:fractions No. 16 and 17; G5 fraction: fractions No.18-21), where theinhibitory activity was observed, were dried using centrifugalevaporator.

[0056] The active fractions obtained by gel filtration were thenfractionated by using reverse column COSMOSIL 5C18 ARA4.6/250 (NakaraiTesque). Solution A (distilled water containing 0.05% TFA) and solutionB (acetonitrile containing 0.065% TFA) was used as the mobile phase, andthe chromatography was carried out using concentration gradienttechnique where the concentration of solution B increased from 0% to 50%for 50 minutes with the flow rate of 0.75 ml/min. 0.75 ml aliquots werecollected. The results showed that the angiotensin conversion enzymeinhibitory activity tend to be high when the concentration gradient ofsolution B is in the range from about 20% to 30% of solution B (FIG. 5).

[0057] These fractions were subjected to MS/MS analysis to determine thesequences of the peptides, which resulted in obtaining 74 candidates forangiotensin converting enzyme inhibitory peptides.

[0058] Among the candidate peptides, five peptides were selected whichwere expected from the sequence to be derived from storage proteinstaking large scale preparation into account, and which contained Proresidue or which were expected to have high hydrophobicvity.

Example 3 Synthesis of the Candidate Peptides as Angiotensin ConvertingEnzyme Inhibitor

[0059] To elucidate whether or not these candidate peptides haveangiotensin converting enzyme inhibitory activity, the peptides weresynthesized by solid phase method. The procedure of synthesizingTyr-Val-Val-Phe-Lys is described below. 50 mg of Fmoc-Lys(Boc) resin(p-alkoxy benzyl alcohol resin into which Fmoc-Lys(Boc)-OH wasintroduced at a ratio of 0.48 mmol/g resin; Nova Biochem) was suspendedin DMF (1 ml) and the suspension was shaken for 1 hour to allow theresin to swell.

[0060] The resin was subjected to the following Fmoc group removingcycle and Fmoc amino acid condensation cycle.

[0061] a) shaking in 1 ml of DMF for 1 minute (once);

[0062] b) shaking in 600 μl of 50% piperidine-DMF solution for 12minutes:

[0063] c) washing 5 times with 600 μl of DMF;

[0064] d) wash once with 1 ml of isopropanol;

[0065] e) allowing the resin obtained at Fmoc group removing cycle toswell by shaking in 1 ml of DMF twice;

[0066] f) Fmoc-Phe-OH (15 mg) and HOBt (8 mg) were dissolved in 800 μland added to the resin, 50 μl of 1M dicyclohexylcarbadiimide methylenechloride solution was then added and was shaken for 60 minutes;

[0067] g) washing twice with 1 ml of DMF; and,

[0068] h) washing twice with isopropanol.

[0069] Fmoc group removing cycle (a-d) and Fmoc amino acid condensationcycle (f-h) were repeated similarly to condense Fmoc-Val-OH, Fmoc-Val-OHand Fmoc-Tyr(But)-OH (all of them were supplied by Nova Biochem)sequentially to obtain Fmoc-Tyr(But)-Val-Val-Phe-Lys(Boc)resin.

[0070] Fmoc group was then removed by treating the protected peptidylresin with 50% piperidine-DMF solution and the product was subjected tothe following deprotection step:

[0071] a) shaking for 12 minutes after adding 1ml of ether;

[0072] b) drying by using centrifugal evaporator;

[0073] c) shaking for 60 minutes after adding 1 ml ofphenol-water-thioanisole-etahnediol-TFA (1.42: 1.4: 1.5: 0.9: 24.9);

[0074] d) filtrating the resin, washing twice the residual resin with 1ml of TFA and combining the filtrate;

[0075] e) adding 10 ml of ether and 1 ml of water to fractionate aqueousphase;

[0076] f) further washing the aqueous phase twice; and,

[0077] g) freeze drying to obtain crude peptide.

[0078] The obtained crude peptide was purified to exhibit a single peakin reverse phase chromatography analysis using lnertsil ODS column (GLScience). Additionally, the results of mass spectrometry analysisconsisted with the theoretical values. The similar reaction and processas described above were carried out for other peptides to synthesize thepeptides shown in Table 1. TABLE 1 Angiotensin converting enzymeinhibitor peptide candidates (Theo- Mass reti- spec- cal Peptidetroscopy value) Tyr-Val-Val-Phe-Lys 655.2 (654.8)Pro-Asn-Asn-Lys-Pro-Phe-Gln 844.6 (843.9) Asn-Trp-Gly-Pro-Leu-Val 685.6(684.8) Ile-Pro-Pro-Gly-Val-Pro-Tyr-Trp-Thr 522.5 (521.6)Thr-Pro-Arg-Val-Phe 655.6 (654.8)

Example 4 Determination of the Inhibitory Activity of AngiotensinConverting Enzyme Inhibitory Peptide Candidates

[0079] To 50 μl of each peptide solution sample, 125 μl of 10 mMp-hydroxyl benzoyl glycyl-L-histidyl-L-leucine, 2.5 mM4-aminoantipyrine, 3 units/ml hipricase (solution in 0.2M borate buffercontaining 0.7M NaCl) were added and pre-incubated at 37° C. for 3minutes. 20 μl of 200 mU/ml rabbit pulmonal angiotensin convertingenzyme (Sigma) was added to the solution to start the reaction. Afterincubating at 37° C. for 20 minutes, the reaction was stopped by adding375 μl of 3 mM EDTA, 0.2% Triton X-100, 6.5 mM of sodium periodatesolution. After incubating for further 3 minutes for color development.The reaction solution was subjected to colorimetry at a wavelength of505 nm. Distilled water was used as a control. The concentrationexhibiting 50% inhibition was indicated in Table 2 as IC₅₀ values. TheIC₅₀ of Bradykinin potentiator C (Peptide Institution) as determined bythis method was shown as the positive control. TABLE 2 Inhibitoryactivity of Angiotensin converting enzyme inhibitory peptide candidatesPeptide IC₅₀ (μM) Tyr-Val-Val-Phe-Lys 44 Pro-Asn-Asn-Lys-Pro-Phe-Gln 33Asn-Trp-Gly-Pro-Leu-Val 21 Ile-Pro-Pro-Gly-Val-Pro-Tyr-Trp-Thr 64Thr-Pro-Arg-Val-Phe 200 Bradykinin potentiator C 35

Example 5 Determination of Hypotensive Activity of D3-DegradationProduct

[0080] (1) Preparation for Hydrolysates with D3

[0081] Isolated soybean protein (AP-SU, Ajinomoto Co., Inc.) weredissolved in 50 ml of deionized water, denatured at 120° C. for 20minutes and adjusted to pH 4.5. D3 was added to 0.5% of the substrateand allowed to react on the substrate at 40° C. for 48 hours. After thereaction was completed the mixture was centrifuged and the pH of thesupernatant was adjusted to about neutral point. The supernatant wasthen heated to 100° C. for 10 minutes to inactivate the enzyme. Aftercooling on ice, desalination was carried out by using an electrodialyzer(Micro Acilyzer G3, Asahikasei, AC-110-800 cartridge); the product wasfreeze-dried and stored. The average molecular weight of the obtainedhydrolysates was 1053.IC₅₀ value, the concentration for 50% inhibitionagainst ACE, was 180 μg/ml as measured according to the same method asdescribed in Example 4.

[0082] (2) Determination of Hypotensive Effect of Hydrolysates

[0083] The 9 weeks old spontaneously hypertensive model rats(SHR/Izm:SPF) were purchase from Japan SLC Co. The rats were pre-fedincluding quarantine of one week. The animals which did not exhibit anyabnormality were used to estimate the hypotensive effect ofhydrolysates. The animals were kept separately in movable stainless rackin the environment of 22±3° C., 50±20% humidity, 12 hours light/darkcycle (8:00-20:00) and 13-17 times/hour ventilation. The animals werefed with solid diet, Labo MR Stock (Nihon Nousan Industry, Co.), bystainless solid diet feeder and, for the water, the animals received tapwater through a polysulphone waterer, and both of them were given adlibitum. The amount of the soybean protein hydrolysates in each groupwas shown in Table 3. The hydrolysates were dissolved in distilled water(Otuka Distilled Water, Otuka Pharmaceutical Co., Ltd.). The controlgroup received only the distilled water. 4 hours after receiving them,the rats were given 9 g of feed, when the measurement was finished.TABLE The amount of hydrolysates given in each group Received amountReceived Volume Number of Group (mg/kg) (ml/kg) animals Control 0 10 7soybean protein 50 10 7 hydrolysates 100 10 7 500 10 7 1000 10 7

[0084] The tests were carried out by determining the blood pressure andheart rate of 10 weeks old SHR rat by using noninvasive automated bloodpressure manometer for small animals (MK-2000, Muromachi Kikai, Co.).Classification was carried out by stratified sequential randomizedcontrolled trial using systolic blood pressure as the indicator. Aftergrouping, the rats were starved overnight, and then the test sampleswere orally injected at 10 ml/(kg body weight). After 0, 1, 2, 4 and 6hours, the blood pressure and heart rate of the rats were determined.The significant difference between each group was determined bycomparing the average values for each group which received the sampleagainst the control group using Tukey-Kramer method (StatView-J 5.0) andthe significant difference was to be recognized when the significancelevel was 5% or less.

[0085] When SHRs received 50, 100, 500 and 1000 mg/(kg body weight) ofsoybean protein hydrolysates, they exhibited the blood pressure decreasestarting at 1 hour after giving, when compared with the control group.Regarding dose response, the statistical significant difference was notobserved between 50 mg/(kg body weight) group and the control group. Thegroup received 100 mg/(kg body weight) exhibited the significantdecrease compared with the control group only at 1 hour after theadministration, and the group received 500 mg/(kg body weight) and 1000mg/(kg body weight) exhibited the significant reduce compared with thecontrol group at 1 and 2 hours after administration. The results wereshown in Table 4 and FIG. 6. TABLE 4 Hypotensive effect of soy proteinhydrolysates Change in blood pressure Hours after administrationHydrolysates 0 1 2 4   0 mg/kg 175.7 ± 3.0 164.5 ± 4.2 161.7 ± 1.6 149.3± 1.8  50 mg/kg 174.8 ± 3.1 152.6 ± 4.1 145.0 ± 7.1 143.5 ± 7.9  100mg/kg 175.5 ± 2.7 150.0 ± 2.8* 140.8 ± 2.6 140.5 ± 6.6  500 mg/kg 176.5± 4.0 148.8 ± 2.7* 134.1 ± 3.8** 146.3 ± 2.2 1000 mg/kg 175.1 ± 3.0140.7 ± 123.7 ± 2.3** 137.6 ± 4.5 2.5** Change in blood pressure Hoursafter administration Hydrolysates 6 8 24   0 mg/kg 162.3 ± 2.7 168.9 ±3.4 182.1 ± 1.2  50 mg/kg 156.0 ± 3.0 163.3 ± 4.9 177.1 ± 1.5  100 mg/kg148.4 ± 4.3 160.9 ± 3.9 178.4 ± 1.1  500 mg/kg 152.4 ± 5.5 162.8 ± 4.7177.9 ± 1.9 1000 mg/kg 148.8 ± 3.1 157.3 ± 4.5 176.3 ± 2.8

[0086] The changes in heart rate in these groups were not observed,although the data were not shown.

[0087] From these data, soybean protein hydrolysate was shown to exhibitsignificant hypotensive effect in vivo at the administration of 100mg/(kg body weight) or more in SHR and the dosage response was observed.

[0088] According to the present invention, biologically active peptides,namely, angiotensin converting enzyme inhibitor peptides, which arederived from soybean protein and which have improved taste, areprovided. Additionally, according to the method of present invention forproducing angiotensin converting enzyme inhibitor peptides, enzymatichydrolysates containing angiotensin converting enzyme inhibitor peptideshaving low bitterness can be produced. These soybean proteinhydrolysates containing these peptides have the hypotensive effect.Thus, the angiotensin converting enzyme inhibitory peptides of thepresent invention and the hydrolysates containing the peptides can beused for various foods including healthy foods having hypotensiveeffect.

1 14 1 5 PRT Glycine max 1 Tyr Val Val Phe Lys 1 5 2 7 PRT Glycine max 2Pro Asn Asn Lys Pro Phe Gln 1 5 3 6 PRT Glycine max 3 Asn Trp Gly ProLeu Val 1 5 4 9 PRT Glycine max 4 Ile Pro Pro Gly Val Pro Tyr Trp Thr 15 5 5 PRT Glycine max 5 Thr Pro Arg Val Phe 1 5 6 1392 DNA Glycine maxmat_peptide (397)..() 6 atg acc atg gct gcg atc gtg ctc ctg ttc acg gtcttt gcc gtt 45 Met Thr Met Ala Ala Ile Val Leu Leu Phe Thr Val Phe AlaVal -130 -125 -120 tcc tcc gcc cta gac atg tcg ata atc tcg tac gac agcgcc cac 90 Ser Ser Ala Leu Asp Met Ser Ile Ile Ser Tyr Asp Ser Ala His-115 -110 -105 gcg gac aag gcc gcc acg ttg cgc acc gag gag gag ctg atgtcc atg 138 Ala Asp Lys Ala Ala Thr Leu Arg Thr Glu Glu Glu Leu Met SerMet -100 -95 -90 tac gag cag tgg ctc gtg aag cac ggg aag gtg tac aac gcgctc ggc 186 Tyr Glu Gln Trp Leu Val Lys His Gly Lys Val Tyr Asn Ala LeuGly -85 -80 -75 gag aag gag aag cgc ttc cag atc ttc aag gac aac ctg cgattc atc 234 Glu Lys Glu Lys Arg Phe Gln Ile Phe Lys Asp Asn Leu Arg PheIle -70 -65 -60 -55 gac gac cac aac tcc gcg gag gac cga acc tac aag ctcgga ctg aac 282 Asp Asp His Asn Ser Ala Glu Asp Arg Thr Tyr Lys Leu GlyLeu Asn -50 -45 -40 cgg ttc gct gat ctc acc aac gag gaa tac agg gcc aagtac ttg gga 330 Arg Phe Ala Asp Leu Thr Asn Glu Glu Tyr Arg Ala Lys TyrLeu Gly -35 -30 -25 acc aag atc gat ccc aac cgg agg ctc gga aag acc ccgagc aac cgc 378 Thr Lys Ile Asp Pro Asn Arg Arg Leu Gly Lys Thr Pro SerAsn Arg -20 -15 -10 tac gcg cca cgt gtc ggc gac aaa ttg cct gat tcc gttgat tgg agg 426 Tyr Ala Pro Arg Val Gly Asp Lys Leu Pro Asp Ser Val AspTrp Arg -5 -1 1 5 10 aag gaa ggt gct gtt cct cct gtc aaa gac caa gga ggctgt ggg agc 474 Lys Glu Gly Ala Val Pro Pro Val Lys Asp Gln Gly Gly CysGly Ser 15 20 25 tgt tgg gca ttc tca gca atc ggt gca gta gaa gga ata aataag ata 522 Cys Trp Ala Phe Ser Ala Ile Gly Ala Val Glu Gly Ile Asn LysIle 30 35 40 gta aca ggc gaa ctg att tcg tta tca gaa caa gaa ttg gtg gattgt 570 Val Thr Gly Glu Leu Ile Ser Leu Ser Glu Gln Glu Leu Val Asp Cys45 50 55 gat act gga tat aac caa gga tgc aat gga gga ctt atg gac tat gca618 Asp Thr Gly Tyr Asn Gln Gly Cys Asn Gly Gly Leu Met Asp Tyr Ala 6065 70 ttt gag ttc ata atc aac aat ggc ggc att gat tct gat gag gat tac666 Phe Glu Phe Ile Ile Asn Asn Gly Gly Ile Asp Ser Asp Glu Asp Tyr 7580 85 90 cca tac cgt ggt gtt gat ggt aga tgc gac aca tat agg aaa aat gct714 Pro Tyr Arg Gly Val Asp Gly Arg Cys Asp Thr Tyr Arg Lys Asn Ala 95100 105 aaa gtc gtt tct att gat gac tac gaa gat gtt cct gcc tat gat gag762 Lys Val Val Ser Ile Asp Asp Tyr Glu Asp Val Pro Ala Tyr Asp Glu 110115 120 tta gcc ttg aaa aag gcc gtt gca aat cag ccc gtg agc gtt gct att810 Leu Ala Leu Lys Lys Ala Val Ala Asn Gln Pro Val Ser Val Ala Ile 125130 135 gaa gga ggg ggc agg gaa ttt caa tta tat gta tct ggt gta ttc acg858 Glu Gly Gly Gly Arg Glu Phe Gln Leu Tyr Val Ser Gly Val Phe Thr 140145 150 ggg aga tgt ggc aca gca cta gat cat ggt gtc gtg gct gtt ggg tat906 Gly Arg Cys Gly Thr Ala Leu Asp His Gly Val Val Ala Val Gly Tyr 155160 165 170 gga aca gct aaa ggt cat gat tat tgg atc gta agg aat tca tggggt 954 Gly Thr Ala Lys Gly His Asp Tyr Trp Ile Val Arg Asn Ser Trp Gly175 180 185 tct agc tgg gga gag gat ggc tac atc aga tta gaa aga aat cttgct 1002 Ser Ser Trp Gly Glu Asp Gly Tyr Ile Arg Leu Glu Arg Asn Leu Ala190 195 200 aac agc aga tca ggc aag tgt gga att gca att gag cca tct tatccc 1050 Asn Ser Arg Ser Gly Lys Cys Gly Ile Ala Ile Glu Pro Ser Tyr Pro205 210 215 ctt aag aat ggt cca aat ccc cct aat cct gga cca tca ccc ccttca 1098 Leu Lys Asn Gly Pro Asn Pro Pro Asn Pro Gly Pro Ser Pro Pro Ser220 225 230 cct gtg aag ccg cca aat gtc tgt gac aac tac tac agc tgt gctgat 1146 Pro Val Lys Pro Pro Asn Val Cys Asp Asn Tyr Tyr Ser Cys Ala Asp235 240 245 250 agt gct act tgt tgc tgt att ttt gag ttc gga aat gct tgcttc gag 1194 Ser Ala Thr Cys Cys Cys Ile Phe Glu Phe Gly Asn Ala Cys PheGlu 255 260 265 tgg ggt tgc tgt cct ctt gag ggt gct tcc tgc tgt gat gaccac tac 1242 Trp Gly Cys Cys Pro Leu Glu Gly Ala Ser Cys Cys Asp Asp HisTyr 270 275 280 agt tgc tgc cct gca gac tat ccc atc tgc aac act tac gctgga act 1290 Ser Cys Cys Pro Ala Asp Tyr Pro Ile Cys Asn Thr Tyr Ala GlyThr 285 290 295 tgt ctc agg agc aag aac aac ccc ttt gga gtg aag gca ttaagg cgt 1338 Cys Leu Arg Ser Lys Asn Asn Pro Phe Gly Val Lys Ala Leu ArgArg 300 305 310 act cca gcg aaa ccc cat tgg acc ttc gga cgt aag aac aaggtc agc 1386 Thr Pro Ala Lys Pro His Trp Thr Phe Gly Arg Lys Asn Lys ValSer 315 320 325 330 agt gct 1392 Ser Ala 7 464 PRT Glycine max 7 Met ThrMet Ala Ala Ile Val Leu Leu Phe Thr Val Phe Ala Val -130 -125 -120 SerSer Ala Leu Asp Met Ser Ile Ile Ser Tyr Asp Ser Ala His -115 -110 -105Ala Asp Lys Ala Ala Thr Leu Arg Thr Glu Glu Glu Leu Met Ser Met -100 -95-90 Tyr Glu Gln Trp Leu Val Lys His Gly Lys Val Tyr Asn Ala Leu Gly -85-80 -75 Glu Lys Glu Lys Arg Phe Gln Ile Phe Lys Asp Asn Leu Arg Phe Ile-70 -65 -60 -55 Asp Asp His Asn Ser Ala Glu Asp Arg Thr Tyr Lys Leu GlyLeu Asn -50 -45 -40 Arg Phe Ala Asp Leu Thr Asn Glu Glu Tyr Arg Ala LysTyr Leu Gly -35 -30 -25 Thr Lys Ile Asp Pro Asn Arg Arg Leu Gly Lys ThrPro Ser Asn Arg -20 -15 -10 Tyr Ala Pro Arg Val Gly Asp Lys Leu Pro AspSer Val Asp Trp Arg -5 -1 1 5 10 Lys Glu Gly Ala Val Pro Pro Val Lys AspGln Gly Gly Cys Gly Ser 15 20 25 Cys Trp Ala Phe Ser Ala Ile Gly Ala ValGlu Gly Ile Asn Lys Ile 30 35 40 Val Thr Gly Glu Leu Ile Ser Leu Ser GluGln Glu Leu Val Asp Cys 45 50 55 Asp Thr Gly Tyr Asn Gln Gly Cys Asn GlyGly Leu Met Asp Tyr Ala 60 65 70 Phe Glu Phe Ile Ile Asn Asn Gly Gly IleAsp Ser Asp Glu Asp Tyr 75 80 85 90 Pro Tyr Arg Gly Val Asp Gly Arg CysAsp Thr Tyr Arg Lys Asn Ala 95 100 105 Lys Val Val Ser Ile Asp Asp TyrGlu Asp Val Pro Ala Tyr Asp Glu 110 115 120 Leu Ala Leu Lys Lys Ala ValAla Asn Gln Pro Val Ser Val Ala Ile 125 130 135 Glu Gly Gly Gly Arg GluPhe Gln Leu Tyr Val Ser Gly Val Phe Thr 140 145 150 Gly Arg Cys Gly ThrAla Leu Asp His Gly Val Val Ala Val Gly Tyr 155 160 165 170 Gly Thr AlaLys Gly His Asp Tyr Trp Ile Val Arg Asn Ser Trp Gly 175 180 185 Ser SerTrp Gly Glu Asp Gly Tyr Ile Arg Leu Glu Arg Asn Leu Ala 190 195 200 AsnSer Arg Ser Gly Lys Cys Gly Ile Ala Ile Glu Pro Ser Tyr Pro 205 210 215Leu Lys Asn Gly Pro Asn Pro Pro Asn Pro Gly Pro Ser Pro Pro Ser 220 225230 Pro Val Lys Pro Pro Asn Val Cys Asp Asn Tyr Tyr Ser Cys Ala Asp 235240 245 250 Ser Ala Thr Cys Cys Cys Ile Phe Glu Phe Gly Asn Ala Cys PheGlu 255 260 265 Trp Gly Cys Cys Pro Leu Glu Gly Ala Ser Cys Cys Asp AspHis Tyr 270 275 280 Ser Cys Cys Pro Ala Asp Tyr Pro Ile Cys Asn Thr TyrAla Gly Thr 285 290 295 Cys Leu Arg Ser Lys Asn Asn Pro Phe Gly Val LysAla Leu Arg Arg 300 305 310 Thr Pro Ala Lys Pro His Trp Thr Phe Gly ArgLys Asn Lys Val Ser 315 320 325 330 Ser Ala 8 357 DNA ArtificialSequence Synthetic DNA 8 cgcccaatac gcaaaccgcc tctccccgcg cgttggccgcttcattaatg cagctggcac 60 gacaggtttc ccgactggaa agcgggcagt gagcgcaacgcaattaatgt gagttagctc 120 actcattagg caccccaggc tttacacttt atgcttccggatcgtatgtt gtgtggaatt 180 gtgagcggat aacaatttca cacaggaaac agctatgaccatgattacgc caagcttgca 240 tgcctgcagg tcgccctttc gtcttcaaga attcccctgttgacaattaa tcatcgaact 300 agttaactag tacgcaagtt cacgtaaaaa gggtatcgattagtaaggag gtttaaa 357 9 20 DNA Artificial Sequence Synthetic DNA 9cgcccaatac gcaaaccgcc 20 10 27 DNA Artificial Sequence Synthetic DNA 10tttaaacctc cttactaatc gataccc 27 11 32 DNA Artificial Sequence SyntheticDNA 11 ggaggtttaa aatgtacgac agcgcccacg cg 32 12 23 DNA ArtificialSequence Synthetic DNA 12 caagcttgta ggttcggtcc tcc 23 13 25 DNAArtificial Sequence Synthetic DNA 13 tacgacagcg cccacgcgga caagg 25 1437 DNA Artificial Sequence Synthetic DNA 14 gggatcctca gctgtagtagttgtcacaga catttgg 37

What is claimed is:
 1. A peptide represented by any of the followingformula (1) to (5) or the salts thereof: (1)Tyr-Val-Val-Phe-Lys;(2)Pro-Asn-Asn-Lys-Pro-Phe-Gln; (3)Asn-Trp-Gly-Pro-Leu-Val;(4)Ile-Pro-Pro-Gly-Val-Pro-Tyr-Trp-Thr; (5)Thr-Pro-Arg-Val-Phe.
 2. Anangiotensin inhibitor comprising the peptides according to claim 1 orthe salts thereof.
 3. The angiotensin inhibitor characterized in that itis a hydrolysate of soybean proteins.
 4. A method of producing theangiotensin inhibitor which comprises hydrolyzing a soybean protein withD3.
 5. An antihypertensive comprising the peptide of claim 1 or thesalts thereof.
 6. A food comprising the peptides according to claim 1 orthe salts thereof.
 7. A method for producing the peptide according toclaim 1 comprising hydrolyzing a soy protein using a protease.
 8. Amethod for producing the peptide according to claim 1 comprisinghydrolyzing a soybean protein with protease D3 derived from germinatingsoybean.